Objective To identify Tyrophagus fanetzhangorum and T. putrescentiae by morphological and molecular methods. Methods Samples were collected from houses and stored products, and individual mites were selected for pure culture. The cultured adult mites were used to prepare slide specimens for morphological identification based on the taxonomic keys of Astigmata. Subsequently, genomic DNA was extracted from individual mites for the PCR amplification, and sequencing of mitochondrial cytochrome c oxidase subunit Ⅰ gene (CO Ⅰ). The resulting sequences were subjected to homology search using BLAST in NCBI. The CO Ⅰ sequences of T. fanetzhangorum and T. putrescentiae were aligned using Clustal Ⅹ 1.83. Sequence analysis was performed using MEGA Ⅹ, and a phylogenetic tree was constructed using the maximum likelihood method. Results The posterior 1/3 of the coxal plate Ⅱ of T. fanetzhangorum was concave, the inferior margin was "S" shaped, and the distal 2/3 of solenidion ω ₁ on tarsus Ⅰ obviously widened. The coxal plate Ⅱ of T. putrescentiae was triangular, the inferior margin was straight or slightly concave, and the solenidion ω ₁ on tarsus Ⅰ widened at the distal 1/4. The CO Ⅰ gene sequences of the isolated T. fanetzhangorum and T. putrescentiae showed >99.00% similarity with the CO Ⅰ gene sequences of T. fanetzhangorum and T. putrescentiae in GenBank. The phylogenetic tree showed that T. fanetzhangorum and T. putrescentiae clustered into a branch with a bootstrap value of 100%. Conclusion Identification of T. fanetzhangorum and T. putrescentiae can be performed based on morphological characteristics or molecular analysis of the COⅠ gene. The combination of morphological and molecular identification can improve the accuracy of the identification of closely related mite species.